LECTURE 1 – MOLECULAR AND CELLULAR BIOLOGY

LECTURE 1 – MOLECULAR AND CELLULAR BIOLOGY What do cells do? - Cell proliferation, specialization, interaction, movement. (universal mechanism of animal development) - Cancer is progressive accumulation of mutations Imaging technologies - Resolving power o Smallest distance you can see two points of an image - Resolution o Lower the wavelength the better o Numerical aperture if objective lens is measure of light gathering capacity of lens- higher the better - Light microscope - Specimen Preparation (fix and section) o Fixing preserves cells within the tissue o Sectioning- thin transparent slices of tissue o Two main techniques § Frozen • Tissue quickly frozen, sectioned in cryostat, fixed and stained § Paraffin Sections • Fixation • Dehydration • Embedding (paraffin wax) • Sectioning (microtome) • Mounted on glass slides • Stained Fluorescence Microscopy - Sample is light source - Florescence by dyes, proteins, antibody-dyes, auto florescence - High intensity light to excite fluorescence - Molecules emit a longer wavelength than absorbed

Light Micro

Fluorescence Microscopy

Fluorescent probes - DAPI gives off blue colour o binds to A-T rich regions of DNA - Immunofluorescence o Coupling florescent dye (fluorescein or rhodamine) to antibody molecule - Green fluorescent protein o GFP from jelly fish o Placed under transcriptional control of promotor of a gene o Peptide location signal to organelles o GFP DNA coding sequence added at end or start Electron Microscopy - Fine structure - Accelerated electrons - Resolution better - Transmission electron microscopy o Fixation § Glutaraldehyde in phosphate or cacodylate buffer o Processing § Post fixation § Dehydration § Infiltration o Embedding § Epon-araldite o Sectioning § Glass or diamond knives § TEM sections thinner than 10 x resin sections for LM and 100 thinner than paraffin § Sections floated off edge into water and picked by metal grids